Brushes were vortexed vigorously, and the harvested cell suspension was filtered through a 200-μm filter (Millipore; Billerica, MA) to remove mucus and cellular debris, and then treated with 4.8 U/mL of neutral protease, grade 2 (Dispase II; Roche; Mannheim, Germany) to eliminate cell clumping. The cells were then centrifuged at 150g for 10 min, and the pellet was resuspended in cell culture medium. Cell number was determined using a hemocytometer, and cell viability was determined by trypan blue exclusion. Cells were resuspended in serum-free bronchial epithelial cell growth medium (Promocell) supplemented with a variety of growth factors, including bovine pituitary extract (0.052 mg/mL), recombinant human epidermal growth factor (0.5 ng/mL), insulin (5 μg/mL), hydrocortisone (0.5 μg/mL), epinephrine (0.5 μg/mL), triiodothyronine (6.5 ng/mL), transferrin (0.01 mg/mL), and retinoic acid (0.1 ng/mL). In addition, the medium contained penicillin G (100 U/mL), streptomycin (100 μg/mL), and amphotericin B (0.25 μg/mL) [Invitrogen; Karlsruhe, Germany]. Cultures were maintained in a humidified atmosphere at 37°C in air/carbon dioxide (95/5% volume/volume); 0.5 × 105 cells were plated on six-well culture dishes and re-fed three times a week until they were confluent. The identity of the epithelial cells was confirmed in all cultures by light microscopy and in randomly selected cultures by immunocytochemical staining for cytokeratin expression using a pancytokeratin antibody (clone KL1) directed against cytokeratin types 1, 2, 5, 6, 7, 8, 11, 14, 16, and 18 (Immunotec; Marl, Germany). A monoclonal antifibroblast antibody (FibAS02; Dianova; Hamburg, Germany) was used to exclude contamination by fibroblasts.12The staining was performed according to Kunz-Schughart and coworkers13 as described in detail previously.